Blood clotting is the mechanism that prevents continuing blood loss when any part of the blood circulation system is injured. It involves the formation of a semi-solid mass of blood material, which acts to plug vessel wounds. The system comprises series of interacting components in the blood and in the vessel wall that must remain in balance; an inactive system causes risk of severe and fatal bleeding, whereas excessive activity of the blood clotting system causes risk of blood clot formation (thrombosis and thromboembolism) within the circulatory system, clogging arteries, with potentially fatal consequences due to tissue necrosis (cell death).
Anticoagulants or “blood thinners” are among the most frequently administered drug classes and within this class, vitamin K antagonists (VKA, coumarins) have until lately been the only available oral agents. Due to the delicate balance of the blood clotting system and much variation in patient sensitivity and responsiveness to the therapy, VKA doses have to be carefully adjusted and continuously monitored, as patient response and suitable dose frequently changes with time. An estimated 800 million tests are performed annually worldwide to monitor the blood clotting in patients on VKA.
The blood coagulation system comprises a complex system of inter-linked proenzymes, enzymes and cofactors, performing their role on the surface of activated platelets and endothelial cells and in ruptured vessels in the human body. When the system is activated, usually in a vessel wound, the ultimate result is the formation of a blood clot containing an insoluble fibrin mesh. In the body, the coagulation process is carefully controlled on the surface of activated platelets and endothelial cells, but in the testing laboratory, the platelet surface is usually substituted with suitable phospholipids. The proenzymes, enzymes and cofactors are traditionally named coagulation factors (F), most of whom are formed in the liver.
Vitamin K dependent coagulation factors and vitamin K antagonists: Four coagulation factors, ie FII, FVII, FIX and FX, that are formed in the liver, are inactive unless following synthesis they become further carboxylated by a vitamin K dependent enzyme process in the liver. In patients deficient in vitamin K or in patients treated with a vitamin K antagonist (VKA), the amount of carboxylated vitamin K dependent (VKD) factors is reduced and, hence, also the clottability (coagulability) of the relevant patient's blood. Unless the effect of VKA is controlled this may lead to severe and even fatal internal hemorrhage. But by reducing the VKD factor levels in a controlled manner, abnormal blood clotting within the vessels (thrombosis) can be prevented while minimizing the risk of bleeding. It is therefore imperative that the effect of VKA be monitored with appropriate coagulation tests. Based on test results, the dose can then be adjusted maximizing the antithrombotic effect and at the same time minimizing the risk of abnormal bleeding caused by over-anticoagulation.
The monitoring of blood clotting activity in VKA patients has for over sixty years been based on measuring the prothrombin time (PT), either as the original Quick PT (PT) (Quick, A., J Bio Chem 1935(109): p. 73-4) or as it's modification, the Owren's PT (also known as PP, P&P-test or prothrombin complex test)(Owren and Aas. Scand J Clin Lab Invest, 1951. 3(3): p. 201-8.). The PT test measures the clotting activity of three out of four vitamin K dependent clotting factors (i.e. FII, FVII, and FX) as well as that of fibrinogen (factor I) and factor V, in a blood plasma sample which has been depleted from calcium, by adding a coagulation reagent (thromboplastin, tissue factor) and calcium, and subsequently measuring the time that it takes the blood to coagulate. The latter test (P&P) is a modification of the PT, where adsorbed plasma (totally deficient in vitamin K dependent factors) is mixed into the test plasma, correcting for any possible deficiency of factor V or fibrinogen and leaving the P&P test only sensitive to factors II, VII and X. Hence, the PT test is also sensitive to deficiency of factors V and fibrinogen, which are not vitamin K dependent and if deficient may confound results in patients taking VKA. With both tests, however, the measured clotting time is equally sensitive to a reduction in any of the three vitamin K dependent factors that the test measures, i.e. FII, FVII and FX. FIX coagulant activity is not measured by these tests. The clotting times obtained with the PT based tests have in practice been presumed to directly reflect the antithrombotic effect of VKA in patients except during initiation of VKA therapy. However, this may not always be the case.
The actual measured clotting time measured on a normal individual will vary, depending on the method applied and also the analytical system and specific reagents used. Different coagulation reagents and even different batches of manufacturers' tissue factor (thromboplastin) and proteins used cause variability in the clotting time obtained with the PT or P&P. Therefore, a protocol for calibrating methods has been devised. Each manufacturer assigns an ISI value (International Sensitivity Index) for the provided tissue factor. (See WHO Guidelines for Thromboplastins and Plasma Used to Control Anticoagulant Therapy, TRS, No 889, Annex 3.) The ISI value indicates how a particular batch of tissue factor compares to an internationally standardized sample. The ISI is usually between 1.0 and 2.0. An “International normalised ratio” value (INR) can thus be calculated, which is the ratio of a patient's prothrombin time to the mean normal population PT, raised to the power of the ISI value for the analytical system used. The INR value can accordingly be described with the equation:INR=(PT/MNPT)ISI where MNPT refers to “mean normal prothrombin time”. For practical purposes the MNPT is generally obtained as a mean value of prothrombin times of at least 20 fresh samples from healthy individuals. A high INR level, such as INR over 5, indicates that there is a high chance of bleeding, whereas if the INR is 1.3 or less, there is no protection against having thromboembolism. A normal INR range for a healthy person is 0.8-1.3 and for people on coumarin therapy (e.g. warfarin), the most commonly recommended therapeutic range is 2.0-3.0. The target INR may be set higher in particular situations, such as for individuals with mechanical heart valves.
Modified tests for accurately assessing blood clottability that could overcome disadvantages of present methods in the art, and make possible more accurate dose adjustment and preferably less frequent monitoring of the anticoagulant activity in anticoagulated patients, would be greatly appreciated.